1. Field of the Invention
The present invention relates to processes which amplify nucleic acid sequences. In particular, the present invention discloses a means for eliminating the products of an execution of a nucleic acid amplification process that contaminate subsequent executions of the amplification process.
2. Description of Related Disclosures
The polymerase chain reaction (PCR) procedure amplifies specific nucleic acid sequences through a series of manipulations including denaturation, annealing of primers, and extension of the primers with DNA polymerase (Mullis KB et al., U.S. Pat. No. 4,683,202, U.S. Pat. No. 4,683,195; Mullis KB, EP 201,184; Erlich H, EP 50,424, EP 84,796, EP 258,017, EP 237,362; Erlich H, U.S. Pat. No. 4,582,788; Saiki R et al., U.S. Pat. No. 4,683,202; Mullis KB et al. (1986) in Cold Spring Harbor Symp. Quant. Biol. 51:263; Saiki R et al. (1985) Science 230:1350; Saiki R et al. (1988) Science 231 487; Loh EY et al. (1988) Science 243:217; etc.). (References cited herein are hereby incorporated by reference.) These steps can be repeated many times, potentially resulting in large amplifications of the number of copies of the original specific sequence. It has been shown that even single molecules of DNA can be amplified to produce hundreds of nanograms of product (Li H et al. (1988) Nature 335:414).
Other known nucleic acid amplification procedures include the transcription-based amplification system of Kwoh D et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173.
"Uracil DNA glycosylase" (UDG), a term of art, refers to an enzyme which cleaves the glycosidic bond between the base uracil and the sugar deoxyribose, only when the monomeric nucleotide dUTP is incorporated into a DNA molecule, resulting in incorporation of a deoxyuridine moiety (Duncan B (1981) in The Enzymes 14:565, ed.: Boyer P). The enzyme does not act upon free dUTP, free deoxyuridine, or RNA (Duncan, supra).
A consequence of amplification processes such as PCR is that the amplification products themselves can be substrates for subsequent PCR procedures. Furthermore, because the quantities of the amplification products can be large, the dispersal of even an extremely small fraction of a reaction such as a PCR reaction into the laboratory area potentially can lead to contamination of later attempts to amplify other samples.
The present invention represents an improvement upon in vitro nucleic acid amplification procedures in general by making amplification products distinguishable from naturally occurring DNA. Accordingly, such products are rendered inactive as templates for further amplification prior to the start of the succeeding amplification reaction.